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  • 351. Sweetlove, Lee J.
    et al.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fernie, Alisdair R.
    Engineering central metabolism - a grand challenge for plant biologists2017In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 4, p. 749-763Article in journal (Refereed)
    Abstract [en]

    The goal of increasing crop productivity and nutrient-use efficiency is being addressed by a number of ambitious research projects seeking to re-engineer photosynthetic biochemistry. Many of these projects will require the engineering of substantial changes in fluxes of central metabolism. However, as has been amply demonstrated in simpler systems such as microbes, central metabolism is extremely difficult to rationally engineer. This is because of multiple layers of regulation that operate to maintain metabolic steady state and because of the highly connected nature of central metabolism. In this review we discuss new approaches for metabolic engineering that have the potential to address these problems and dramatically improve the success with which we can rationally engineer central metabolism in plants. In particular, we advocate the adoption of an iterative 'design-build-test-learn' cycle using fast-to-transform model plants as test beds. This approach can be realised by coupling new molecular tools to incorporate multiple transgenes in nuclear and plastid genomes with computational modelling to design the engineering strategy and to understand the metabolic phenotype of the engineered organism. We also envisage that mutagenesis could be used to fine-tune the balance between the endogenous metabolic network and the introduced enzymes. Finally, we emphasise the importance of considering the plant as a whole system and not isolated organs: the greatest increase in crop productivity will be achieved if both source and sink metabolism are engineered.

  • 352. Szatmári, T.
    et al.
    Mundt, F.
    Heidari-Hamedani, G.
    Zong, F.
    Ferolla, E.
    Alexeyenko, Andrey
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hjerpe, A.
    Dobra, K.
    Novel Genes and Pathways Modulated by Syndecan-1: Implications for the Proliferation and Cell-Cycle Regulation of Malignant Mesothelioma Cells2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10, p. e48091-Article in journal (Refereed)
    Abstract [en]

    Malignant pleural mesothelioma is a highly malignant tumor, originating from mesothelial cells of the serous cavities. In mesothelioma the expression of syndecan-1 correlates to epithelioid morphology and inhibition of growth and migration. Our previous data suggest a complex role of syndecan-1 in mesothelioma cell proliferation although the exact underlying molecular mechanisms are not completely elucidated. The aim of this study is therefore to disclose critical genes and pathways affected by syndecan-1 in mesothelioma; in order to better understand its importance for tumor cell growth and proliferation. We modulated the expression of syndecan-1 in a human mesothelioma cell line via both overexpression and silencing, and followed the transcriptomic responses with microarray analysis. To project the transcriptome analysis on the full-dimensional picture of cellular regulation, we applied pathway analysis using Ingenuity Pathway Analysis (IPA) and a novel method of network enrichment analysis (NEA) which elucidated signaling relations between differentially expressed genes and pathways acting via various molecular mechanisms. Syndecan-1 overexpression had profound effects on genes involved in regulation of cell growth, cell cycle progression, adhesion, migration and extracellular matrix organization. In particular, expression of several growth factors, interleukins, and enzymes of importance for heparan sulfate sulfation pattern, extracellular matrix proteins and proteoglycans were significantly altered. Syndecan-1 silencing had less powerful effect on the transcriptome compared to overexpression, which can be explained by the already low initial syndecan-1 level of these cells. Nevertheless, 14 genes showed response to both up- and downregulation of syndecan-1. The "cytokine - cytokine-receptor interaction", the TGF-β, EGF, VEGF and ERK/MAPK pathways were enriched in both experimental settings. Most strikingly, nearly all analyzed pathways related to cell cycle were enriched after syndecan-1 silencing and depleted after syndecan-1 overexpression. Syndecan-1 regulates proliferation in a highly complex way, although the exact contribution of the altered pathways necessitates further functional studies.

  • 353. Sánchez, J. L. A.
    et al.
    Henry, O. Y. F.
    Joda, H.
    Solnestam, Beata Werne
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Erik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lladach, N.
    Ramakrishnan, D.
    Riley, I.
    O'Sullivan, C. K.
    Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach2016In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 82, p. 224-232Article in journal (Refereed)
    Abstract [en]

    Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25 pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".

  • 354. Tellgren-Roth, Asa
    et al.
    Dittmar, Katharina
    Massey, Steven E.
    Kemi, Cecilia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Tellgren-Roth, Christian
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lyons, Leslie A.
    Liberles, David A.
    Keeping the blood flowing-plasminogen activator genes and feeding behavior in vampire bats2009In: Die Naturwissenschaften, ISSN 0028-1042, E-ISSN 1432-1904, Vol. 96, no 1, p. 39-47Article in journal (Refereed)
    Abstract [en]

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  • 355. Tepeli, Cafer
    et al.
    Erdoğan, Metin
    Yılmaz, Alper
    Bulut, Zafer
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Some morphological characteristics of Turkish shepherd dogs raised in breeder conditions various countries: Çeşitli ülkelerde halk elinde yetiştirilen Türk Çoban Köpeklerinde bazı morfolojik özellikler2017In: Eurasian Journal of Veterinary Sciences, ISSN 1309-6958, Vol. 33, no 4, p. 268-275Article in journal (Refereed)
    Abstract [en]

    Aim: This research was conducted to compare some morphological characteristics of shepherd dogs, phenotypically similar to Turkish shepherd dogs, raised in public areas in Turkey, Iran, Azerbaijan and Uzbekistan.

    Materials and Methods: A total of 468 dogs of different ages were used in the study. Body measurements such as shoulder height, rump height, body length, front chest width, chest depth, chest girth, head girth, head length and muzzle length were taken from the dogs.

    Results: There were significant differences between Kangal dogs in Turkey and dogs in the Kangal phenotype of Iran, Azerbaijan and Uzbekistan regarding some body measurements, such as shoulder height,, body length, and chest circumference (P<0.01). It can be said that Kangal dogs raised in public areas in Turkey are bigger size than Kangal dogs in other countries.

    Conclusion: Morphological comparisons of Turkish Shepherd Dog Breeds can be extended by including similar phenotype dogs in Turkmenistan, Kyrgyzstan and Afghanistan.

  • 356.
    The, Matthew
    KTH, School of Biotechnology (BIO), Gene Technology.
    Statistical and machine learning methods to analyze large-scale mass spectrometry data2016Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    As in many other fields, biology is faced with enormous amounts ofdata that contains valuable information that is yet to be extracted. The field of proteomics, the study of proteins, has the luxury of having large repositories containing data from tandem mass-spectrometry experiments, readily accessible for everyone who is interested. At the same time, there is still a lot to discover about proteins as the main actors in cell processes and cell signaling.

    In this thesis, we explore several methods to extract more information from the available data using methods from statistics and machine learning. In particular, we introduce MaRaCluster, a new method for clustering mass spectra on large-scale datasets. This method uses statistical methods to assess similarity between mass spectra, followed by the conservative complete-linkage clustering algorithm.The combination of these two resulted in up to 40% more peptide identifications on its consensus spectra compared to the state of the art method.

    Second, we attempt to clarify and promote protein-level false discovery rates (FDRs). Frequently, studies fail to report protein-level FDRs even though the proteins are actually the entities of interest. We provided a framework in which to discuss protein-level FDRs in a systematic manner to open up the discussion and take away potential hesitance. We also benchmarked some scalable protein inference methods and included the best one in the Percolator package. Furthermore, we added functionality to the Percolator package to accommodate the analysis of studies in which many runs are aggregated. This reduced the run time for a recent study regarding a draft human proteome from almost a full day to just 10 minutes on a commodity computer, resulting in a list of proteins together with their corresponding protein-level FDRs.

  • 357.
    The, Matthew
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    MaRaCluster: A Fragment Rarity Metric for Clustering Fragment Spectra in Shotgun Proteomics2016In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 15, no 3, p. 713-720Article in journal (Refereed)
    Abstract [en]

    Shotgun proteomics experiments generate large amounts of fragment spectra as primary data, normally with high redundancy between and within experiments. Here, we have devised a clustering technique to identify fragment spectra stemming from the same species of peptide. This is a powerful alternative method to traditional search engines for analyzing spectra, specifically useful for larger scale mass spectrometry studies. As an aid in this process, we propose a distance calculation relying on the rarity of experimental fragment peaks, following the intuition that peaks shared by only a few spectra offer more evidence than peaks shared by a large number of spectra. We used this distance calculation and a complete-linkage scheme to cluster data from a recent large-scale mass spectrometry-based study. The clusterings produced by our method have up to 40% more identified peptides for their consensus spectra compared to those produced by the previous state-of-the-art method. We see that our method would advance the construction of spectral libraries as well as serve as a tool for mining large sets of fragment spectra. The source code and Ubuntu binary packages are available at https://github.com/ statisticalbiotechnology/maracluster (under an Apache 2.0 license).

  • 358.
    The, Matthew
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    MacCoss, M. J.
    Noble, W. S.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fast and Accurate Protein False Discovery Rates on Large-Scale Proteomics Data Sets with Percolator 3.02016In: Journal of the American Society for Mass Spectrometry, ISSN 1044-0305, E-ISSN 1879-1123, Vol. 27, no 11, p. 1719-1727Article in journal (Refereed)
    Abstract [en]

    Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator’s processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore, with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method—grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein—in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 million spectra (PM:24870542). The source code and Ubuntu, Windows, MacOS, and Fedora binary packages are available from http://percolator.ms/ under an Apache 2.0 license. [Figure not available: see fulltext.]

  • 359.
    The, Matthew
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    MacCoss, Michael J.
    Noble, William S.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology.
    Fast and accurate protein false discovery rates on large-scale proteomics data sets with Percolator 3.0Manuscript (preprint) (Other academic)
    Abstract [en]

    Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator's processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore,with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method - grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein - in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 million spectra (PM:24870542).

    The source code and Ubuntu, Windows, MacOS and Fedora binary packages are available from http://percolator.ms/ under an Apache 2.0 license.

  • 360.
    The, Matthew
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Tasnim, Ayesha
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology.
    How to talk about protein-level false discovery rates in shotgun proteomicsManuscript (preprint) (Other academic)
    Abstract [en]

    A frequently sought output from a shotgun proteomics experiment is a list of proteins that we believe to have been present in the analyzed sample before proteolytic digestion. The standard technique to control for errors in such lists is to enforce a preset threshold for the false discovery rate. Many researchers consider protein-level false discovery rates a difficult and vague concept, as the measurement entities, spectra, are manifestations of peptides and not proteins. Here, we argue that this confusion is unnecessary and provide a framework on how to think about protein-level false discovery rates, starting from its basic principle: the null hypothesis. Specifically, we point out that two competing null hypotheses are used concurrently in today's protein inference methods, which has gone unnoticed by many. Using simulations of a shotgun proteomics experiment, we show how confusing one null hypothesis for the other can lead to serious discrepancies in the false discovery rate. Furthermore, we demonstrate how the same simulations can be used to verify false discovery rate estimates of protein inference methods. In particular, we show that, for a simple protein inference method, decoy models can be used to accurately estimate protein-level false discovery rates for both competing null hypotheses.

  • 361.
    The, Matthew
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Tasnim, Ayesha
    KTH, School of Biotechnology (BIO), Gene Technology.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology.
    How to talk about protein-level false discovery rates in shotgun proteomics2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 18, p. 2461-2469Article in journal (Refereed)
    Abstract [en]

    A frequently sought output from a shotgun proteomics experiment is a list of proteins that we believe to have been present in the analyzed sample before proteolytic digestion. The standard technique to control for errors in such lists is to enforce a preset threshold for the false discovery rate (FDR). Many consider protein-level FDRs a difficult and vague concept, as the measurement entities, spectra, are manifestations of peptides and not proteins. Here, we argue that this confusion is unnecessary and provide a framework on how to think about protein-level FDRs, starting from its basic principle: the null hypothesis. Specifically, we point out that two competing null hypotheses are used concurrently in today's protein inference methods, which has gone unnoticed by many. Using simulations of a shotgun proteomics experiment, we show how confusing one null hypothesis for the other can lead to serious discrepancies in the FDR. Furthermore, we demonstrate how the same simulations can be used to verify FDR estimates of protein inference methods. In particular, we show that, for a simple protein inference method, decoy models can be used to accurately estimate protein-level FDRs for both competing null hypotheses.

  • 362. Thureborn, P.
    et al.
    Lundin, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Plathan, J.
    Poole, A. M.
    Sjöberg, B. -M
    Sjöling, S.
    A Metagenomics Transect into the Deepest Point of the Baltic Sea Reveals Clear Stratification of Microbial Functional Capacities2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e74983-Article in journal (Refereed)
    Abstract [en]

    The Baltic Sea is characterized by hyposaline surface waters, hypoxic and anoxic deep waters and sediments. These conditions, which in turn lead to a steep oxygen gradient, are particularly evident at Landsort Deep in the Baltic Proper. Given these substantial differences in environmental parameters at Landsort Deep, we performed a metagenomic census spanning surface to sediment to establish whether the microbial communities at this site are as stratified as the physical environment. We report strong stratification across a depth transect for both functional capacity and taxonomic affiliation, with functional capacity corresponding most closely to key environmental parameters of oxygen, salinity and temperature. We report similarities in functional capacity between the hypoxic community and hadal zone communities, underscoring the substantial degree of eutrophication in the Baltic Proper. Reconstruction of the nitrogen cycle at Landsort deep shows potential for syntrophy between archaeal ammonium oxidizers and bacterial denitrification at anoxic depths, while anaerobic ammonium oxidation genes are absent, despite substantial ammonium levels below the chemocline. Our census also reveals enrichment in genetic prerequisites for a copiotrophic lifestyle and resistance mechanisms reflecting adaptation to prevalent eutrophic conditions and the accumulation of environmental pollutants resulting from ongoing anthropogenic pressures in the Baltic Sea.

  • 363. Ting, Ying S.
    et al.
    Egertson, Jarrett D.
    Payne, Samuel H.
    Kim, Sangtae
    MacLean, Brendan
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aebersold, Ruedi
    Smith, Richard D.
    Noble, William Stafford
    MacCoss, Michael J.
    Peptide-Centric Proteome Analysis: An Alternative Strategy for the Analysis of Tandem Mass Spectrometry Data2015In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, no 9, p. 2301-2307Article, review/survey (Refereed)
    Abstract [en]

    In mass spectrometry-based bottom-up proteomics, data-independent acquisition is an emerging technique because of its comprehensive and unbiased sampling of precursor ions. However, current data-independent acquisition methods use wide precursor isolation windows, resulting in cofragmentation and complex mixture spectra. Thus, conventional database searching tools that identify peptides by interpreting individual tandem MS spectra are inherently limited in analyzing data-independent acquisition data. Here we discuss an alternative approach, peptide-centric analysis, which tests directly for the presence and absence of query peptides. We discuss how peptide-centric analysis resolves some limitations of traditional spectrum-centric analysis, and we outline the unique characteristics of peptide-centric analysis in general.

  • 364. Torkzaban, Bahareh
    et al.
    Kayvanjoo, Amir Hossein
    Ardalan, Arman
    KTH, School of Biotechnology (BIO), Gene Technology. Natl Inst Genet Engn & Biotechnol, Iran.
    Mousavi, Soraya
    Mariotti, Roberto
    Baldoni, Luciana
    Ebrahimie, Esmaeil
    Ebrahimi, Mansour
    Hosseini-Mazinani, Mehdi
    Machine Learning Based Classification of Microsatellite Variation: An Effective Approach for Phylogeographic Characterization of Olive Populations2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 11, article id e0143465Article in journal (Refereed)
    Abstract [en]

    Finding efficient analytical techniques is overwhelmingly turning into a bottleneck for the effectiveness of large biological data. Machine learning offers a novel and powerful tool to advance classification and modeling solutions in molecular biology. However, these methods have been less frequently used with empirical population genetics data. In this study, we developed a new combined approach of data analysis using microsatellite marker data from our previous studies of olive populations using machine learning algorithms. Herein, 267 olive accessions of various origins including 21 reference cultivars, 132 local ecotypes, and 37 wild olive specimens from the Iranian plateau, together with 77 of the most represented Mediterranean varieties were investigated using a finely selected panel of 11 microsatellite markers. We organized data in two '4-targeted' and '16-targeted' experiments. A strategy of assaying different machine based analyses (i.e. data cleaning, feature selection, and machine learning classification) was devised to identify the most informative loci and the most diagnostic alleles to represent the population and the geography of each olive accession. These analyses revealed microsatellite markers with the highest differentiating capacity and proved efficiency for our method of clustering olive accessions to reflect upon their regions of origin. A distinguished highlight of this study was the discovery of the best combination of markers for better differentiating of populations via machine learning models, which can be exploited to distinguish among other biological populations.

  • 365. Tuominen, Rainer
    et al.
    Engstrom, Par G.
    Helgadottir, Hildur
    Eriksson, Hanna
    Unneberg, Per
    Kjellqvist, Sanela
    Yang, Muyi
    Linden, Diana
    Edsgärd, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hansson, Johan
    Hoiom, Veronica
    The Role of Germline Alterations in the DNA Damage Response Genes BRIP1 and BRCA2 in Melanoma Susceptibility2016In: Genes, Chromosomes and Cancer, ISSN 1045-2257, E-ISSN 1098-2264, Vol. 55, no 7, p. 601-611Article in journal (Refereed)
    Abstract [en]

    We applied a targeted sequencing approach to identify germline mutations conferring a moderately to highly increased risk of cutaneous and uveal melanoma. Ninety-two high-risk melanoma patients were screened for inherited variation in 120 melanoma candidate genes. Observed gene variants were filtered based on frequency in reference populations, cosegregation with melanoma in families and predicted functional effect. Several novel or rare genetic variants in genes involved in DNA damage response, cell-cycle regulation and transcriptional control were identified in melanoma patients. Among identified genetic alterations was an extremely rare variant (minor allele frequency of 0.00008) in the BRIP1 gene that was found to cosegregate with the melanoma phenotype. We also found a rare nonsense variant in the BRCA2 gene (rs11571833), previously associated with cancer susceptibility but not with melanoma, which showed weak association with melanoma susceptibility in the Swedish population. Our results add to the growing knowledge about genetic factors associated with melanoma susceptibility and also emphasize the role of DNA damage response as an important factor in melanoma etiology.

  • 366. Uddenberg, Daniel
    et al.
    Reimegård, Johan
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Clapham, David
    Almqvist, Curt
    von Arnold, Sara
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sundström, Jens F.
    Early Cone Setting in Picea abies acrocona Is Associated with Increased Transcriptional Activity of a MADS Box Transcription Factor2013In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 161, no 2, p. 813-823Article in journal (Refereed)
    Abstract [en]

    Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A(+)) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce.

  • 367.
    Uhlén, Mathias
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lindskog, Cecilia
    Mardinoglu, Adil
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Pontén, Fredrik
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark; Chalmers University of Technology, Sweden.
    Transcriptomics resources of human tissues and organs2016In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 12, no 4, article id 862Article, review/survey (Refereed)
    Abstract [en]

    Quantifying the differential expression of genes in various human organs, tissues, and cell types is vital to understand human physiology and disease. Recently, several large-scale transcriptomics studies have analyzed the expression of protein-coding genes across tissues. These datasets provide a framework for defining the molecular constituents of the human body as well as for generating comprehensive lists of proteins expressed across tissues or in a tissue-restricted manner. Here, we review publicly available human transcriptome resources and discuss body-wide data from independent genome-wide transcriptome analyses of different tissues. Gene expression measurements from these independent datasets, generated using samples from fresh frozen surgical specimens and postmortem tissues, are consistent. Overall, the different genome-wide analyses support a distribution in which many proteins are found in all tissues and relatively few in a tissue-restricted manner. Moreover, we discuss the applications of publicly available omics data for building genome-scale metabolic models, used for analyzing cell and tissue functions both in physiological and in disease contexts.

  • 368. Unneberg, Per
    et al.
    Stromberg, Michael
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Jansson, Stefan
    Sterky, Fredrik
    KTH, School of Biotechnology (BIO), Proteomics.
    Analysis of 70,000 EST sequences to study divergence between two closely related Populus species2005In: Tree Genetics & Genomes, ISSN 1614-2942, Vol. 1, no 3, p. 109-115Article in journal (Refereed)
    Abstract [en]

    The Populus genus has evolved as the model organism for forest tree genomics, which has been further emphasised with the sequencing of the Populus trichocarpa genome. Populus species are widely spread over the Northern Hemisphere and provide a great source of genetic diversity, which can be used for mapping of quantitative trait loci, positional cloning, association mapping and studies in environmental adaptation. Collections of expressed sequence tags (ESTs) are rich sources in studies of genetic diversity. Here, we report on an in-depth analysis of 70,000 ESTs from two Populus species, Populus tremula and Populus trichocarpa. We present data on the level of conservation in transcript sequences and supply a collection of potential single nucleotide polymorphisms.

  • 369. van Asch, Barbara
    et al.
    Zhang, Ai-bing
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Oskarsson, Mattias C. R.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Klütsch, Cornelya F. C.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Amorim, Antonio
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pre-Columbian origins of Native American dog breeds, with only limited replacement by European dogs, confirmed by mtDNA analysis2013In: Proceedings of the Royal Society of London. Biological Sciences, ISSN 0962-8452, E-ISSN 1471-2954, Vol. 280, no 1766, p. 20131142-Article in journal (Refereed)
    Abstract [en]

    Dogs were present in pre-Columbian America, presumably brought by early human migrants from Asia. Studies of free-ranging village/street dogs have indicated almost total replacement of these original dogs by European dogs, but the extent to which Arctic, North and South American breeds are descendants of the original population remains to be assessed. Using a comprehensive phylogeographic analysis, we traced the origin of the mitochondrial DNA lineages for Inuit, Eskimo and Greenland dogs, Alaskan Malamute, Chihuahua, xoloitzcuintli and perro sin pelo del Peru, by comparing to extensive samples of East Asian (n = 984) and European dogs (n = 639), and previously published pre-Columbian sequences. Evidence for a pre-Columbian origin was found for all these breeds, except Alaskan Malamute for which results were ambigous. No European influence was indicated for the Arctic breeds Inuit, Eskimo and Greenland dog, and North/South American breeds had at most 30% European female lineages, suggesting marginal replacement by European dogs. Genetic continuity through time was shown by the sharing of a unique haplotype between the Mexican breed Chihuahua and ancient Mexican samples. We also analysed free-ranging dogs, confirming limited pre-Columbian ancestry overall, but also identifying pockets of remaining populations with high proportion of indigenous ancestry, and we provide the first DNA-based evidence that the Carolina dog, a free-ranging population in the USA, may have an ancient Asian origin.

  • 370. van Asch, Barbara
    et al.
    Zhang, Ai-bing
    KTH, School of Biotechnology (BIO), Gene Technology.
    Oskarsson, Mattias
    KTH, School of Biotechnology (BIO), Gene Technology.
    Klütsch, Cornelya
    KTH, School of Biotechnology (BIO), Gene Technology.
    Amorim, António
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    MtDNA analysis confirms early Pre‐Colombian origins of Native AmericandogsManuscript (preprint) (Other academic)
    Abstract [en]

    Dogs were present in Pre‐Columbian America, presumably brought to the New World by early human migrants of Asian origin. However, the extent to which historical Arctic, North and South American breeds, e.g. the Alaskan Malamute, Inuit, Eskimo and Greenland dogs, Xoloitzcuintli, Chihuahua and Perro Sín Pelo del Peru, are descendants of these original dogs or were replaced by European dogs remains to be assessed. Using a comprehensive phylogeographic analysis to trace the origin of their mitochondrial (mt) DNA lineages, these breeds were analysed for 582 bp of the mtDNA control region and compared to extensive samples of East Asian (n = 984) and European dogs (n = 639), and previously published Pre‐Columbian sequences. Evidence for a Pre‐Columbian origin was found for all putative American breeds based on the detection of haplotypes phylogenetically distinct from European haplotypes, exclusive to America, shared only with East Asia, or identical to ancient American sequences. Identical rare haplotypes in ancient and modern samples showed geographic continuity over time in Mexico (Chihuahua) and Alaska (Alaskan Malamute). A European origin for at most 15% of the female lineages was indicated, suggesting marginal replacement by European dogs. We also analysed free‐ranging dogs from the USA and South America. In agreement with a previous study, free ranging dogs in general showed little pre‐Columbian ancestry. However, for several populations an appreciable proportion of indigenous ancestry was indicated. Specifically, we provide the first DNA‐based evidence for an ancient Asian origin of the Carolina Dog, a dingo‐like free‐ranging population in the USA. Numerous dogs were probably brought from Asia, since totally 13 mtDNA haplotypes among extant and ancient American dogs were distinct from haploypes found in Europe.

  • 371. van der Oost, John
    et al.
    Walther, Jasper
    Brouns, Stan J. J.
    van de Verken, Harmen J. G.
    Snijders, Ambrosius P. L.
    Wright, Phillip C.
    Andersson, Anders
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Bernander, Rolf
    de Vos, Willem M.
    Functional genomics of the thermo-acidophilic archaeon Sulfolobus solfataricus2006In: EXTREMOPHILES / [ed] Rainey, FA; Oren, A, LONDON: ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD , 2006, Vol. 35, p. 201-231Chapter in book (Other academic)
  • 372. van Rooijen, Marianne
    et al.
    Silveira, Angela
    Thomassen, Stella
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Gene Technology.
    Hamsten, Anders
    Rosing, Jan
    Bremme, Katarina
    APC resistance during the normal menstrual cycle2007In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 98, no 6, p. 1246-1251Article in journal (Refereed)
    Abstract [en]

    Increased serum levels of endogenous as well as exogenous estrogen are regarded to be responsible for acquired activated protein C (APC) resistance. It was the objective of this study to evaluate whether the physiological increase in serum estradiol concentration during the normal menstrual cycle affects the individual's sensitivity to APC. Seventy-two women with normal menstrual cycles were included in the study. Blood samples for analysis of estradiol (E2), progesterone (P4) and APC resistance were drawn at two time points of the menstrual cycle (day 3-5 and day 22-25). Two methods of measuringAPC resistance were used: the activated partial thromboplastin time (aPTT)-based assay and the endogenous thrombin potential (ETP)-basedAPC resistance test. Independent of the method used, no changes in APC resistance were found, even though the E2 concentration increased significantly between the two menstrual phases. No correlations between E2 levels andAPC resistance, P4 levels and APC resistance or changes in E2 concentrations and changes in APC resistance were detected. Ten women were carriers of the factor V-Leiden mutation, Their baseline APC resistance was increased, but their response to elevated E2 during the menstrual cycle did not differ from that of non-carriers. In conclusion, our observations suggest that physiological differences in serum levels of estradiol and progesterone between the early follicular and the luteal phase in a normal menstrual cycle do not have any significant impact on the individual's sensitivity to APC.

  • 373. Varemo, Leif
    et al.
    Henriksen, Tora Ida
    Scheele, Camilla
    Broholm, Christa
    Pedersen, Maria
    Uhlen, Mathias
    Pedersen, Bente Klarlund
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Type 2 diabetes and obesity induce similar transcriptional reprogramming in human myocytes2017In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 9, article id 47Article in journal (Refereed)
    Abstract [en]

    Background: Skeletal muscle is one of the primary tissues involved in the development of type 2 diabetes (T2D). The close association between obesity and T2D makes it difficult to isolate specific effects attributed to the disease alone. Therefore, here we set out to identify and characterize intrinsic properties of myocytes, associated independently with T2D or obesity. Methods: We generated and analyzed RNA-seq data from primary differentiated myotubes from 24 human subjects, using a factorial design (healthy/T2D and non-obese/obese), to determine the influence of each specific factor on genome-wide transcription. This setup enabled us to identify intrinsic properties, originating from muscle precursor cells and retained in the corresponding myocytes. Bioinformatic and statistical methods, including differential expression analysis, gene-set analysis, and metabolic network analysis, were used to characterize the different myocytes. Results: We found that the transcriptional program associated with obesity alone was strikingly similar to that induced specifically by T2D. We identified a candidate epigenetic mechanism, H3K27me3 histone methylation, mediating these transcriptional signatures. T2D and obesity were independently associated with dysregulated myogenesis, down-regulated muscle function, and up-regulation of inflammation and extracellular matrix components. Metabolic network analysis identified that in T2D but not obesity a specific metabolite subnetwork involved in sphingolipid metabolism was transcriptionally regulated. Conclusions: Our findings identify inherent characteristics in myocytes, as a memory of the in vivo phenotype, without the influence from a diabetic or obese extracellular environment, highlighting their importance in the development of T2D.

  • 374.
    Vazin, Tandis
    KTH, School of Biotechnology (BIO), Gene Technology.
    Generation of Dopaminergic Neurons from Human Embryonic Stem Cells2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Since the first successful derivation of human embryonic stem cells (hESC), rapid progress has been attained in the development of strategies in differentiation of these cells into various neural lineages, with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system (CNS). Of particular interest are midbrain dopaminergic (mDA) neurons, which play a central role in regulation of voluntary movement. Degeneration or loss of function of mDA neurons in the nigrostriatal pathway is associated with Parkinson disease (PD).

    Stromal-Derived Inducing Activity (SDIA) is recognized as one of the most efficient methods in restricting ESC differentiation to a dopaminergic lineage, and refers to the property of mouse stromal cell lines such as PA6 or MS5 to cause ESC to differentiate to DA neurons. Although this strategy has been extensively used to generate mDA neurons from hESC, the biochemical nature of SDIA is yet unknown. 

    In the present study mDA neurons were generated from the BG01V2 hESC line by SDIA. To examine whether SDIA exerts its effect directly on hESC and is responsible for early dopaminergic induction, neural progenitor cells (NPC) were enyzmatically isolated from the co-cultures and allowed to differentiate in feeder-free conditions. The isolated cells were committed to a mesencephalic neural lineage, and were capable of maintaining their phenotype and developing into postmitotic mDA neurons in feeder-free conditions. The mDA neurons showed neuronal excitability and dopamine transporter function. The in vitro proliferation and differentiation of the NPC was also investigated by a BrDU incorporation assay.

    Next, the maintenance of cellular memory and capacity for proliferation of the mesencephalic NPC was assessed. The NPC could be expanded in vitro by five-fold as neurospheres for up to two weeks while retaining their DA differentiation potential, but did not retain a stable phenotype over extended periods of time. Preliminary transplantation experiments of neurospheres in striatal lesioned animals indicated, however, that these cells could survive and conserve their phenotype in vivo.

    To gain additional insight into the biochemical role of SDIA in early dopaminergic induction of hESC, the separate contributions of cell surface activity and secreted factors were examined. The data revealed that the PA6 cell surface activity promoted cell survival and was mainly responsible for enhanced neurogenesis of hESC, whereas secreted factors provided DA lineage-specific instructions.

    In order to identify the soluble factors responsible for the DA phenotype-inducing component of SDIA, the gene expression profile of PA6 cells was compared to that of cell lines lacking the DA-inducing property. A number of soluble factors known to be associated with CNS development that were highly expressed in PA6 cells were identified as potential DA differentiation-inducing candidates. These differentially-expressed genes included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these factors, termed SPIE, were applied to the hESC, they induced dopaminergic neuronal differentiation of hESC line, BG01V2 and other karyotypically normal hESC lines in vitro. Thus, it appears that SPIE comprises the DA phenotype-inducing property of SDIA. This may provide a simple and direct means of differentiating hESC to form DA neurons in a single step, without a requirement for co-culture, animal cell lines, or animal products.

  • 375.
    Vazin, Tandis
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Becker, Kevin G.
    Zhang, Yongqing
    Worden, Lila
    Freed, William J.
    The Molecular Nature of Stromal-Derived Inducing Activity in Dopaminergic Differentiation of Human Embryonic Stem CellsManuscript (Other academic)
    Abstract [en]

    The utilization of Stromal-Derived Inducing Activity (SDIA) is one of the most efficientmethods in generating dopaminergic (DA) neurons from embryonic stem cells (ESC).Neuronal and dopaminergic induction can be achieved by co-culturing ESC with mousestromal cell lines, PA6 or MS5. Although it is clear that SDIA has neural inducing andmidbrain patterning activity, the molecular nature of SDIA is so far unknown. There arecontrary reports that either cell surface activity or factors secreted by PA6 cells areresponsible for SDIA. Recently, we found that PA6 cell surface and extracellular matrixmolecules primarily promoted cell survival and general neurogenesis of hESC. Incontrast, factors secreted by PA6 cells provided lineage-specific instructions in thepresence of a stabilizing factor, heparin. In the present study, in an attempt to identify thefactors responsible for dopaminergic induction of hESC, we performed cell wholegenome expression analysis to search for soluble factors produced by PA6 cells bycomparing them to various cell lines lacking the SDIA effect. Among the soluble secretedfactors differentially expressed by PA6 cells were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1(EFNB1). The combination of these factors, which we termed SPIE, was sufficient toproduce dopaminergic neuronal differentiation of the hESC line BG01V2 and otherkaryotypically normal hESC lines in vitro.

  • 376.
    Vazin, Tandis
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Chen, J.
    Lee, C. -T
    Amable, R.
    Freed, W. J.
    Assessment of stromal-derived inducing activity in generation of dopaminergic neurons from human embryonic stem cells2007In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 16, no 3, p. 349-349Article in journal (Other academic)
  • 377.
    Vazin, Tandis
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Chen, Jia
    National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD.
    Lee, Chun-Ting
    National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD.
    Amable, Rose
    National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD.
    Freed, William J.
    National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, MD.
    Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells2008In: Stem Cells, ISSN 1066-5099, E-ISSN 1549-4918, Vol. 26, no 6, p. 1517-1525Article in journal (Refereed)
    Abstract [en]

    Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by β-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect.

  • 378.
    Vazin, Tandis
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Chen, Jia
    Natl Inst Drug Abuse, Dev & Plast Sect, Cellular Neurobiol Res Branch, Intramural Res Program,NIH,Dept Hlth & Human Serv, Baltimore, MD.
    Spivak, Charles E.
    Natl Inst Drug Abuse, Cellular Neurophysiol Sect, Cellular Neurobiol Res Branch, Intramural Res Program,NIH,Dept Hlth & Human Serv, Baltimore, MD .
    Amable, Rose
    Natl Inst Drug Abuse, Dev & Plast Sect, Cellular Neurobiol Res Branch, Intramural Res Program,NIH,Dept Hlth & Human Serv, Baltimore, MD.
    Gabitzsch, Emily
    Natl Inst Drug Abuse, Dev & Plast Sect, Cellular Neurobiol Res Branch, Intramural Res Program,NIH,Dept Hlth & Human Serv, Baltimore, MD.
    Lee, Chun-Ting
    Natl Inst Drug Abuse, Dev & Plast Sect, Cellular Neurobiol Res Branch, Intramural Res Program,NIH,Dept Hlth & Human Serv, Baltimore, MD.
    Lupica, Carl R.
    Natl Inst Drug Abuse, Cellular Neurophysiol Sect, Cellular Neurobiol Res Branch, Intramural Res Program,NIH,Dept Hlth & Human Serv, Baltimore, MD .
    Freed, William J.
    Natl Inst Drug Abuse, Dev & Plast Sect, Cellular Neurobiol Res Branch, Intramural Res Program,NIH,Dept Hlth & Human Serv, Baltimore, MD.
    Dopaminergic neurons derived from BG01V2: a variant of human embryonic stem cell line BG012008In: Restorative Neurology and Neuroscience, ISSN 0922-6028, E-ISSN 1878-3627, Vol. 26, no 6, p. 447-458Article in journal (Refereed)
    Abstract [en]

     Background and Purpose: Human embryonic stem cells (hESC) areconsidered a renewable source of dopamine producing neurons, and are of particularinterest for their potential clinical use in Parkinson’s disease. In this study, wecharacterized human dopaminergic neurons generated by stromal-derived inducingactivity (SDIA) from BG01V2, a strain of human embryonic stem cell line, BG01,characterized by a chromosome 17 trisomy. Similar chromosomal changes have beenrepeatedly observed in hESC cultures in different laboratories, indicating the importanceof chromosome 17 for growth and adaptation of hESC to culture. Methods: Weinvestigated in vitro proliferation of differentiating cells using a BrDU incorporationassay, and monitored the cell population in long term cultures. Despite the cytogeneticabnormality, TH+ neurons were postmitotic at all stages of differentiation. After 30 daysof differentiation, cell division ceased in 91% of the overall population of cells in theculture, indicating intact cell cycle regulation. Results: Expression of midbrain specificmarker genes (Otx2, Pax5, Msx-1) showed differentiation of hESC-derived neuralprogenitor cells into midbrain specific dopamine neurons. These neurons expressed thedopamine transporter (DAT), and displayed functional DAT activity and electricalexcitability. Conclusions: TH+ cells derived from the BG01V2 hESC line using SDIAare postmitotic and have functional characteristics of normal dopaminergic neurons.

  • 379.
    Vazin, Tandis
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Gabitzsch, Emily
    Wang, Yun
    Chen, Jia
    Harvey, Brandon K.
    Worden, Lila
    Freed, William J.
    Limited proliferation capacity of mesencepahlic neural progenitor cells derived from human embryonic stem cellsManuscript (Other academic)
    Abstract [en]

    Midbrain neural progenitor cells (NPC) derived from human embryonic stem cells(hESC) may be useful for development of novel transplantation and gene deliverystrategies. One of the major goals of human transplantation research has been to developa means of generating dopaminergic (DA) neurons in vitro, which can be employed fortransplantation. NPC can be generated from hESC using a number of strategies. Thephenotypic conservation, stability, and differentiation of NPC generated under variousconditions are, however, not well understood. In the present study we generatedexpandable mesencephalic-restricted human NPC from the hESC line BG01V2 under theinfluence of stromal-derived inducing activity (SDIA), and assessed their capacity forproliferation and maintenance of cellular memory. The NPC could be expanded by fivefoldas neurospheres for up to 2 weeks in vitro while retaining their DA differentiationpotential, without a substantial loss of cellular memory and viability. Although cellswere continuously maintained under the influence of the midbrain patterning factors SHHand FGF8, they progressively lost their ability to differentiate to DA neurons andmaintain a stable phenotype in vitro. Preliminary transplantation experiments ofneurospheres with midbrain identity in intrastriatal 6-hydroxydopamine lesioned animalsindicated, however, that these cells could survive and conserve their phenotype in vivo.Therefore, in vitro propagation of SDIA-derived NPC under the present conditions resultsin a gradual loss of growth capacity and multipotency over time, whereas the phenotypeof transplanted NPC remains unaltered.

  • 380. Velazquez-Fernandez, D.
    et al.
    Laurell, Cecilia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Saqui-Salces, M.
    Pantoja, J. P.
    Candanedo-Gonzalez, F.
    Reza-Albarran, A.
    Gamboa-Dominguez, A.
    Herrera, M. F.
    Differential RNA expression profile by cDNA microarray in sporadic primary hyperparathyroidism (pHPT): Primary parathyroid hyperplasia versus adenoma2006In: World Journal of Surgery, ISSN 0364-2313, E-ISSN 1432-2323, Vol. 30, no 5, p. 705-713Article in journal (Refereed)
    Abstract [en]

    Background:Differential diagnosis between adenoma and hyperplasia in primary hyperparathyroidism (pHPT) remains a dilemma. The aim of this study was to assess differences in transcriptional genomic expression profiles between sporadic (nonfamilial) parathyroid hyperplasia (SPH), adenoma, and normal tissue. Methods: Parathyroid tissue from 12 patients with parathyroid adenoma, 3 with SPH, and 2 with normal glands was selected for analysis. Histopathology was reviewed in all cases, and all patients with adenomas presented normocalcemia for a minimum of 6 months after one gland resection. Hybridizations were performed in a microarray containing 19,968 human cDNA clones including contiguous replicates. Direct comparisons were performed with reverse labeling for every different pooled sample entity. Expression levels were analyzed using the SAM, SMA, LIMMA, Cluster, and PAM packages in the R environment for statistical computing. Results: There were significant statistical differences between SPH and adenomas. In the direct comparison, a total of 200 genes showed differential expression (P < 0.03): 61 genes were upregulated (> 1.65-fold increase) and 139 were downregulated (> 1.58-fold decrease) with a B value > 4.68 (99.08% probability of real differential expression). When SPH was compared to normal parathyroid tissue, 50 genes were differentially expressed: 42 were upregulated (> 1.89) and 8 were downregulated (> 1.7) with a B > 4.26 (98.6% probability of real differential expression). At least 17 genes were differentially expressed and able to discriminate SPH from adenoma or normal tissue. Upregulated genes were related to apoptosis inhibition, cell proliferation, transcriptional activity and cell adhesion, among other activities. Downregulated genes were mainly related to ion channel activity, lipopolysaccharides, prostaglandin-d synthase, and integral membrane proteins. Conclusions: Our data suggest that SPH and adenoma have a singular molecular signature that, theoretically, could be used for the differential diagnosis of these entities and normal parathyroid tissue.

  • 381. Velazquez-Fernandez, David
    et al.
    Laurell, Cecilia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Geli, Janos
    Höög, Anders
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Gene Technology.
    Kjellman, Magnus
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Hamberger, Bertil
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Bäckdahl, Martin
    Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy.2005In: Surgery, ISSN 0039-6060, E-ISSN 1532-7361, Surgery, Vol. 138, no 6, p. 1087-1094Article in journal (Refereed)
    Abstract [en]

    Background. Distinguishing between adrenocortical adenomas and carcinomas is often difficult. Our aim was to investigate the differences in transcriptional profiles between benign and malignant adrenocortical neoplasms using complementary DNA microarray techniques.

    Methods. We studied 7 patients with adrenocortical carcinomas and 13 with adenomas. Histopathology was reviewed in all patients, clinical follow-up was at least 1 year. Hybridizations were Performed in duplicate against RNA reference. Expression levels were analyzed in the R environment for statistical computing with the use of aroma, limma, statistics, and class packages.

    Results. Transcriptional profiles were homogeneous among adenomas, while carcinomas were much more heterogeneous. Hierarchical clustering and self-organizing maps could separate clearly carcinomas from adenomas. Among genes that were most significantly upregulated in carcinomas were 2 ubiquilin-related genes (USP4 and UFD1L) and several insulinlike growth factor-related genes (IGF2, IGF2R, IGFBP3 and IGFBP6). Among genes that were most significantly downregulated in carcinomas were a cylokine gene (CXCL10), several genes related to cell metabolism (RARRES2, ALDH1A1, CYBRD1 and GSTA4), and the cadherin 2 gene (CDH2).

    Conclusions. Through the use of cDNA arrays, adrenocortical adenomas and carcinomas appear to be clearly distinguishable on the basis of their specific molecular signature. The biologic importance of the up- and downregulated genes is yet to be determined.

  • 382.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Three-dimensional whole transcriptome analysis of tissue for classification of breast cancer2017Manuscript (preprint) (Other academic)
  • 383.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Three-dimensional whole transcriptome analysis of tissue for classification of breast cancer. Submitted manuscript.2017Manuscript (preprint) (Other academic)
  • 384.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Transcriptome-wide analysis in cells and tissues2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    High-throughput sequencing has greatly influenced the amount of data produced and biological questions asked and answered. Sequencing approaches have also enabled rapid development of related technological fields such as single-cell and spatially resolved expression profiling. The introductory parts of this thesis give an overview of the basic molecular and technological apparatus needed to analyse the transcriptome in cells and tissues. This is succeeded by a summary of present investigations that report recent advancements in RNA profiling.

    RNA integrity needs to be preserved for accurate gene expression analysis. A method providing a low-cost alternative for RNA preservation was reported. Namely, a low concentration of buffered formaldehyde was used for fixation of human cell lines and peripheral blood cells (Paper I). The results from bulk RNA sequencing confirmed gene expression was not negatively impacted with the preservation procedure (r2>0.88) and that long-term storage of such samples was possible (r2=0.95). However, it is important to note that a small population of cells overexpressing a limited amount of genes can skew bulk gene expression analyses making them sufficient only in carefully designed studies. Therefore, gene expression should be investigated at the single cell resolution when possible. A method for high-throughput single cell expression profiling termed microarrayed single-cell sequencing was developed (Paper II). The method incorporated fluorescence-activated cell sorting, sample deposition and profiling of thousands of barcoded single cells in one reaction. After sample attachment to a barcoded array, a high-resolution image was taken which linked the position of each array barcode sequence to each individual deposited cell. The cDNA synthesis efficiency was estimated at 17.3% while detecting 27,427 transcripts per cell on average. Additionally, spatially resolved analysis is important in cell differentiation, organ development and pathological changes. Current methods are limited in terms of throughput, cost and time. For that reason, the spatial transcriptomics method was developed (Paper III). Here, the barcoded microarray was used to obtain spatially resolved expression profiles from tissue sections using the same imaging principle. The mouse olfactory bulb was profiled on a whole-transcriptome scale and the results showed that the expression correlated well (r2=0.94-0.97) as compared to bulk RNA sequencing. The method was 6.9% efficient, reported signal diffusion at ~2 μm and accurately deconvoluted layer-specific transcripts in an unbiased manner. Lastly, the spatial transcriptomics concept was applied to profile human breast tumours in three dimensions (Paper IV). Unbiased clustering revealed previously un-annotated regions and classified them as parts of the immune system, providing a detailed view into complex interactions and crosstalk in the whole tissue volume. Spatial tumour classification divulged that certain parts of the tumour clearly classified as other subtypes as compared to bulk analysis providing useful data for current practice diagnostics.

    The last part of the thesis discusses a look towards the future, how the presented methods could be used, improved upon or combined in translational research.

  • 385.
    Vickovic, Sanja
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lewensohn, Rolf
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Toward Rare Blood Cell Preservation for RNA Sequencing2015In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 17, no 4, p. 352-359Article in journal (Refereed)
    Abstract [en]

    Cancer is driven by various events Leading to cell differentiation and disease progression. Molecular tools are powerful approaches for describing how and why these events occur. With the growing field of next-generation DNA sequencing, there is an increasing need for high-quality nucleic acids derived from human cells and tissues a prerequisite for successful cell profiting. Although advances in RNA preservation have been made, some of the largest biobanks still do not employ RNA blood preservation as standard because of Limitations in low blood-input volume and RNA stability over the whole gene body. Therefore, we have developed a robust protocol for blood preservation and tong-term storage while maintaining RNA integrity. Furthermore, we explored the possibility of using the protocol for preserving rare cell samples, such as circulating tumor cells. The results of our study confirmed that gene expression was not impacted by the preservation procedure (r(2) > 0.88) or by Long-term storage (r(2) = 0.95), with RNA integrity number values averaging over 8. Similarly, cell surface antigens were still available for antibody selection (r(2) = 0.95). Lastly, data mining for fusion events showed that it was possible to detect rare tumor cells among a background of other cells present in blood irrespective of fixation. Thus, the developed protocol would be suitable for rare blood cell preservation followed by RNA sequencing analysis.

  • 386.
    Vickovic, Sanja
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stahl, Patrik L.
    Salmen, Fredrik
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Giatrellis, Sarantis
    Westholm, Jakub Orzechowski
    Mollbrink, Annelie
    Navarro, Jose Fernandez
    Custodio, Joaquin
    Bienko, Magda
    Sutton, Lesley-Ann
    Rosenquist, Richard
    Frisen, Jonas
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Massive and parallel expression profiling using microarrayed single-cell sequencing2016In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, article id 13182Article in journal (Refereed)
    Abstract [en]

    Single-cell transcriptome analysis overcomes problems inherently associated with averaging gene expression measurements in bulk analysis. However, single-cell analysis is currently challenging in terms of cost, throughput and robustness. Here, we present a method enabling massive microarray-based barcoding of expression patterns in single cells, termed MASC-seq. This technology enables both imaging and high-throughput single-cell analysis, characterizing thousands of single-cell transcriptomes per day at a low cost (0.13 USD/cell), which is two orders of magnitude less than commercially available systems. Our novel approach provides data in a rapid and simple way. Therefore, MASC-seq has the potential to accelerate the study of subtle clonal dynamics and help provide critical insights into disease development and other biological processes.

  • 387. Väremo, Leif
    et al.
    Scheele, Camilla
    Broholm, Christa
    Mardinoglu, Adil
    Kampf, Caroline
    Asplund, Anna
    Nookaew, Intawat
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pedersen, Bente Klarlund
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. Chalmers University of Technology, Sweden.
    Proteome- and Transcriptome-Driven Reconstruction of the Human Myocyte Metabolic Network and Its Use for Identification of Markers for Diabetes2015In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 11, no 6, p. 921-933Article in journal (Refereed)
    Abstract [en]

    Skeletal myocytes are metabolically active and susceptible to insulin resistance and are thus implicated in type 2 diabetes (T2D). This complex disease involves systemic metabolic changes, and their elucidation at the systems level requires genome-wide data and biological networks. Genome-scale metabolic models (GEMs) provide a network context for the integration of high-throughput data. We generated myocyte-specific RNA-sequencing data and investigated their correlation with proteome data. These data were then used to reconstruct a comprehensive myocyte GEM. Next, we performed a meta-analysis of six studies comparing muscle transcription in T2D versus healthy subjects. Transcriptional changes were mapped on the myocyte GEM, revealing extensive transcriptional regulation in T2D, particularly around pyruvate oxidation, branched-chain amino acid catabolism, and tetrahydrofolate metabolism, connected through the downregulated dihydrolipoamide dehydrogenase. Strikingly, the gene signature underlying this metabolic regulation successfully classifies the disease state of individual samples, suggesting that regulation of these pathways is a ubiquitous feature of myocytes in response to T2D.

  • 388. Wampach, Linda
    et al.
    Heintz-Buschart, Anna
    Hogan, Angela
    Muller, Emilie E. L.
    Narayanasamy, Shaman
    Laczny, Cedric C.
    Hugerth, Luisa
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bindl, Lutz
    Bottu, Jean
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    de Beaufort, Carine
    Wilmes, Paul
    Colonization and Succession within the Human Gut Microbiome by Archaea, Bacteria, and Microeukaryotes during the First Year of Life2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 738Article in journal (Refereed)
    Abstract [en]

    Perturbations to the colonization process of the human gastrointestinal tract have been suggested to result in adverse health effects later in life. Although much research has been performed on bacterial colonization and succession, much less is known about the other two domains of life, archaea, and eukaryotes. Here we describe colonization and succession by bacteria, archaea and microeukaryotes during the first year of life (samples collected around days 1, 3, 5, 28, 150, and 365) within the gastrointestinal tract of infants delivered either vaginally or by cesarean section and using a combination of quantitative real-time PCR as well as 16S and 18S rRNA gene amplicon sequencing. Sequences from organisms belonging to all three domains of life were detectable in all of the collected meconium samples. The microeukaryotic community composition fluctuated strongly over time and early diversification was delayed in infants receiving formula milk. Cesarean section-delivered (CSD) infants experienced a delay in colonization and succession, which was observed for all three domains of life. Shifts in prokaryotic succession in CSD infants compared to vaginally delivered (VD) infants were apparent as early as days 3 and 5, which were characterized by increased relative abundances of the genera Streptococcus and Staphylococcus, and a decrease in relative abundance for the genera Bifidobacterium and Bacteroides. Generally, a depletion in Bacteroidetes was detected as early as day 5 postpartum in CSD infants, causing a significantly increased Firmicutes/Bacteroidetes ratio between days 5 and 150 when compared to VD infants. Although the delivery mode appeared to have the strongest influence on differences between the infants, other factors such as a younger gestational age or maternal antibiotics intake likely contributed to the observed patterns as well. Our findings complement previous observations of a delay in colonization and succession of CSD infants, which affects not only bacteria but also archaea and microeukaryotes. This further highlights the need for resolving bacterial, archaeal, and microeukaryotic dynamics in future longitudinal studies of microbial colonization and succession within the neonatal gastrointestinal tract.

  • 389. Wang, Guo-Dong
    et al.
    Peng, Min-Sheng
    Yang, He-Chuan
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zhang, Ya-Ping
    Questioning the evidence for a Central Asian domestication origin of dogs2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 19, p. E2554-E2555Article in journal (Refereed)
  • 390. Wang, Guo-dong
    et al.
    Zhai, Weiwei
    Yang, He-chuan
    Fan, Ruo-xi
    Cao, Xue
    Zhong, Li
    Wang, Lu
    Liu, Fei
    Wu, Hong
    Cheng, Lu-guang
    Poyarkov, Andrei D.
    Poyarkov, Nikolai A., Jr.
    Tang, Shu-sheng
    Zhao, Wen-ming
    Gao, Yun
    Lv, Xue-mei
    Irwin, David M.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Wu, Chung-I
    Zhang, Ya-ping
    The genomics of selection in dogs and the parallel evolution between dogs and humans2013In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4, p. 1860-Article in journal (Refereed)
    Abstract [en]

    The genetic bases of demographic changes and artificial selection underlying domestication are of great interest in evolutionary biology. Here we perform whole-genome sequencing of multiple grey wolves, Chinese indigenous dogs and dogs of diverse breeds. Demographic analysis show that the split between wolves and Chinese indigenous dogs occurred 32,000 years ago and that the subsequent bottlenecks were mild. Therefore, dogs may have been under human selection over a much longer time than previously concluded, based on molecular data, perhaps by initially scavenging with humans. Population genetic analysis identifies a list of genes under positive selection during domestication, which overlaps extensively with the corresponding list of positively selected genes in humans. Parallel evolution is most apparent in genes for digestion and metabolism, neurological process and cancer. Our study, for the first time, draws together humans and dogs in their recent genomic evolution.

  • 391. Wang, Guo-Dong
    et al.
    Zhai, Weiwei
    Yang, He-Chuan
    Wang, Lu
    Zhong, Li
    Liu, Yan-Hu
    Fan, Ruo-Xi
    Yin, Ting-Ting
    Zhu, Chun-Ling
    Poyarkov, Andrei D.
    Irwin, David M.
    Hytonen, Marjo K.
    Lohi, Hannes
    Wu, Chung-I
    Savolainen, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Zhang, Ya-Ping
    Out of southern East Asia: the natural history of domestic dogs across the world2016In: Cell Research, ISSN 1001-0602, E-ISSN 1748-7838, Vol. 26, no 1, p. 21-33Article in journal (Refereed)
    Abstract [en]

    The origin and evolution of the domestic dog remains a controversial question for the scientific community, with basic aspects such as the place and date of origin, and the number of times dogs were domesticated, open to dispute. Using whole genome sequences from a total of 58 canids (12 gray wolves, 27 primitive dogs from Asia and Africa, and a collection of 19 diverse breeds from across the world), we find that dogs from southern East Asia have significantly higher genetic diversity compared to other populations, and are the most basal group relating to gray wolves, indicating an ancient origin of domestic dogs in southern East Asia 33 000 years ago. Around 15 000 years ago, a subset of ancestral dogs started migrating to the Middle East, Africa and Europe, arriving in Europe at about 10 000 years ago. One of the out of Asia lineages also migrated back to the east, creating a series of admixed populations with the endemic Asian lineages in northern China before migrating to the New World. For the first time, our study unravels an extraordinary journey that the domestic dog has traveled on earth.

  • 392.
    Werne Solnestam, Beata
    KTH, School of Biotechnology (BIO), Gene Technology.
    Interpreting the human transcriptome2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The human body is made of billions of cells and nearly all have the same genome. However, there is a high diversity of cells, resulted from what part of the genome the cells use, i.e. which RNA molecules are expressed. Rapid advances within the field of sequencing allow us to determine the RNA molecules expressed in a specific cell at a certain time. The use of the new technologies has expanded our view of the human transcriptome and increased our understanding of when, where, and how each RNA molecule is expressed.

    The work presented in this thesis focuses on analysis of the human transcriptome. In Paper I, we describe an automated approach for sample preparation. This protocol was compared with the standard manual protocol, and we demonstrated that the automated version outperformed the manual process in terms of sample throughput while maintaining high reproducibility. Paper II addresses the impact of nuclear transcripts on gene expression. We compared total RNA from whole cells and from cytoplasm, showing that transcripts with long, structured 3’- and 5’-untranslated regions and transcripts with long protein coding sequences tended to be retained in the nucleus. This resulted in increased complexity of the total RNA fraction and fewer reads per unique transcript. Papers III and IV describe dynamics of the human muscle transcriptome. For Paper III, we systematically investigated the transcriptome and found remarkably high tissue homogeneity, however a large number of genes and isoforms were differentially expressed between genders. Paper IV describes transcriptome differences in response to repeated training. No transcriptome-based memory was observed, however a large number of isoforms and genes were affected by training. Paper V describes a transcript profiling protocol based on the method Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification. We designed the method for a few selected transcripts whose expression patterns are important for detecting breast cancer cells, and optimized the method for single cell analysis. We successfully detected cells in human blood samples and applied the method to single cells, confirming the heterogeneity of a cell population.

  • 393.
    Werne Solnestam, Beata
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hällman, Jimmie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs2012In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 13, no 1, p. 574-Article in journal (Refereed)
    Abstract [en]

    Background: The majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. In this study, mRNA fractions from the cytoplasm and from whole cells (total RNA) were prepared from three human cell lines and sequenced using massive parallel sequencing. Results: For all three cell lines, of about 15000 detected genes approximately 400 to 1400 genes were detected in different amounts in the cytoplasmic and total RNA fractions. Transcripts detected at higher levels in the total RNA fraction had longer coding sequences and higher number of miRNA target sites. Transcripts detected at higher levels in the cytoplasmic fraction were shorter or contained shorter untranslated regions. Nuclear retention of transcripts and mRNA degradation via miRNA pathway might contribute to this differential detection of genes. The consequence of the differential detection was further investigated by comparison to proteomics data. Interestingly, the expression profiles of cytoplasmic and total RNA correlated equally well with protein abundance levels indicating regulation at a higher level. Conclusions: We conclude that expression levels derived from the total RNA fraction be regarded as an appropriate estimate of the amount of mRNAs present in a given cell population, independent of the coding sequence length or UTRs.

  • 394. Wiegand, Stephan
    et al.
    Meier, Doreen
    Seehafer, Carsten
    Malicki, Marek
    Hofmann, Patrick
    Schmith, Anika
    Winckler, Thomas
    Foeldesi, Balint
    Boesler, Benjamin
    Nellen, Wolfgang
    Reimegård, Johan
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hällman, Jimmie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Avesson, Lotta
    Söderbom, Fredrik
    Hammann, Christian
    The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 5, p. 3330-3345Article in journal (Refereed)
    Abstract [en]

    Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC(-) strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC(-) strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5' and 3' directions.

  • 395.
    Williams, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO), Gene Technology.
    Meletis, Konstantinos
    Wikström, Lilian
    Carlsson, Leif
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Catalog of gene expression in adult neural stem cells and their in vivo microenvironment2006In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, no 10, p. 1798-1812Article in journal (Refereed)
    Abstract [en]

     Stem cells generally reside in a stem cell micro environment, where cues for self-renewal and differentiation are present. However, the genetic program underlying stem cell proliferation and multipotency is poorly understood. Transcriptome analysis of stem cells and their in vivo microenvironment is one way of uncovering the unique sternness properties and provides a framework for the elucidation of stem cell function. Here, we characterize the gene expression profile of the in vivo neural stem cell microenvironment in the lateral ventricle wall of adult mouse brain and of in vitro proliferating neural stem cells. We have also analyzed an Lhx2-expressing hematopoietic-stem-cell-like cell line in order to define the transcriptome of a well-characterized and pure cell population with stem cell characteristics. We report the generation, assembly and annotation of 50,792 high-quality 5'-end expressed sequence tag sequences. We further describe a shared expression of 1065 transcripts by all three stem cell libraries and a large overlap with previously published gene expression signatures for neural stem/progenitor cells and other multipotent stem cells. The sequences and cDNA clones obtained within this framework provide a comprehensive resource for the analysis of genes in adult stem cells that can accelerate future stem cell research.

  • 396. Willing, Ben P.
    et al.
    Dicksved, Johan
    Halfvarson, Jonas
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lucio, Marianna
    Zheng, Zongli
    Jarnerot, Gunnar
    Tysk, Curt
    Jansson, Janet K.
    Engstrand, Lars
    A Pyrosequencing Study in Twins Shows That Gastrointestinal Microbial Profiles Vary With Inflammatory Bowel Disease Phenotypes2010In: Gastroenterology, ISSN 0016-5085, E-ISSN 1528-0012, Vol. 139, no 6, p. 1844-U105Article in journal (Refereed)
    Abstract [en]

    BACKGROUND & AIMS: The composition of the gastrointestinal microbiota is thought to have an important role in the etiology of inflammatory bowel diseases (IBDs) such as Crohn's disease (CD) and ulcerative colitis (UC). Interindividual variation and an inability to detect less abundant bacteria have made it difficult to correlate specific bacteria with disease. METHODS: We used 454 pyrotag sequencing to determine the compositions of microbial communities in feces samples collected from a cohort of 40 twin pairs who were concordant or discordant for CD or UC, and in mucosal samples from a subset of the cohort. The cohort primarily comprised patients who were in remission, but also some with active disease. RESULTS: The profiles of the microbial community differed with disease phenotypes; relative amounts of bacterial populations correlated with IBD phenotypes. The microbial compositions of individuals with CD differed from those of healthy individuals, but were similar between healthy individuals and individuals with UC. Profiles from individuals with CD that predominantly involved the ileum differed from those with CD that predominantly involved the colon; several bacterial populations increased or decreased with disease type. Changes specific to patients with ileal CD included the disappearance of core bacteria, such as Faecalibacterium and Roseburia, and increased amounts of Enterobacteriaceae and Ruminococcus gnavus. CONCLUSIONS: Bacterial populations differ in abundance among individuals with different phenotypes of CD. Specific species of bacteria are associated with ileal CD; further studies should investigate their role in pathogenesis.

  • 397. Wright, J. C.
    et al.
    Collins, M. O.
    Yu, L.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brosch, M.
    Choudhary, J. S.
    Enhanced peptide identification by electron transfer dissociation using an improved mascot percolator2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 8, p. 478-491Article in journal (Refereed)
    Abstract [en]

    Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a postsearch algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.

  • 398. Wu, Xiaofen
    et al.
    Pedersen, Karsten
    Edlund, Johanna
    Eriksson, Lena
    Astrom, Mats
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Bertilsson, Stefan
    Dopson, Mark
    Potential for hydrogen-oxidizing chemolithoautotrophic and diazotrophic populations to initiate biofilm formation in oligotrophic, deep terrestrial subsurface waters2017In: Microbiome, ISSN 0026-2633, E-ISSN 2049-2618, Vol. 5, article id 37Article in journal (Refereed)
    Abstract [en]

    Background: Deep terrestrial biosphere waters are separated from the light-driven surface by the time required to percolate to the subsurface. Despite biofilms being the dominant form of microbial life in many natural environments, they have received little attention in the oligotrophic and anaerobic waters found in deep bedrock fractures. This study is the first to use community DNA sequencing to describe biofilm formation under in situ conditions in the deep terrestrial biosphere. Results: In this study, flow cells were attached to boreholes containing either "modern marine" or "old saline" waters of different origin and degree of isolation from the light-driven surface of the earth. Using 16S rRNA gene sequencing, we showed that planktonic and attached populations were dissimilar while gene frequencies in the metagenomes suggested that hydrogen-fed, carbon dioxide-and nitrogen-fixing populations were responsible for biofilm formation across the two aquifers. Metagenome analyses further suggested that only a subset of the populations were able to attach and produce an extracellular polysaccharide matrix. Initial biofilm formation is thus likely to be mediated by a few bacterial populations which were similar to Epsilonproteobacteria, Deltaproteobacteria, Betaproteobacteria, Verrucomicrobia, and unclassified bacteria. Conclusions: Populations potentially capable of attaching to a surface and to produce extracellular polysaccharide matrix for attachment were identified in the terrestrial deep biosphere. Our results suggest that the biofilm populations were taxonomically distinct from the planktonic community and were enriched in populations with a chemolithoautotrophic and diazotrophic metabolism coupling hydrogen oxidation to energy conservation under oligotrophic conditions.

  • 399. Yosef, Nir
    et al.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology.
    From sequence to structure to networks2008In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 9, no 11Article in journal (Refereed)
    Abstract [en]

    A report on the 7th European Conference on Computational Biology (ECCB), Cagliari, Italy, 22-26 September 2008.

  • 400. Yu, Tao
    et al.
    Zhou, Yongjin J.
    Wenning, Leonie
    Liu, Quanli
    Krivoruchko, Anastasia
    Siewers, Verena
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    David, Florian
    Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 15587Article in journal (Refereed)
    Abstract [en]

    Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C22H46O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l(-1) in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.

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